We have isolated in purity greater than 98% two cardiotonic polypeptides, anthopleurin-A (AP-A and anthopleurin-C (AP-C) from two species of sea anemones in the genes anthopleura. Since l974 the pharmacology and chemical structures have been thoroughly studied particularly with AP-A. However, a third cardiotonic polypeptide, AP-B, was isolated by similar procedures and thought to be pure until sequence studies and very recent exploratory high pressure liquid chromatography separations indicated AP-B was only about 35% pure. Sephadex retardation indicates the molecular weight of AP-B to be about 2500 daltons instead of approximately 5000 for AP-A and AP-C, with the same cardiotonic potency. The aims of this project are to prepare analytically pure AP-B using HPLC in quantity sufficient to obtain accurate amino acid analyses, molecular weight by high cross-link gel SDS electrophoresis and sephadex G-25 retardation, isoelectric point by gel isoelectric focusing, Rf values in acidic and basic gel electrophoresis, sequence and disulfide cross-link location in collaboration with Dr. K. Yasunobu (U.H.), 13C-NMR secondary structure studies in collaboration with Dr. R.S. Norton (U.N.S.W., Australia), X-ray crystallographic studies by Dr. C.D. Stout (U. Pittsburgh), synthesis of AP-B by Dr. G. Matsueda (Mass. Gen. Hosp.), all of these using methodology the same or similar to that already employed on AP-A. In addition we will supply AP-B to pharmacologists who wish to study the mechanism of positive inotropic action, efficacy and toxicology. I am particularly anxious for Dr. Watanabe at Toyobo Co., Ltd., in Osaka, Japan, to attempt to modify AP-B to prevent potential antigenicity as he is now doing with AP-A. We will enzymatically fragment pure AP-B to see if smaller moieties in AP-B might maintain cardiotonic properties. Activity will be assayed using isolated bath mouse atrial preparation. The long term objective is to provide a superior cardiotonic agent for acute parental and hopefully chronci oral human use, as well as for mechanism of cardiac function studies.